CN110530853B - Method for detecting aflatoxin B1 based on visual BPE-ECL technology - Google Patents
Method for detecting aflatoxin B1 based on visual BPE-ECL technology Download PDFInfo
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- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention discloses a method for detecting aflatoxin B1 based on a visual BPE-ECL technology, which comprises the following steps: 1) preparing a screen printing bipolar electrode; 2) constructing a functional sensing interface; 3) mixing AFB1 with unknown concentration and HRP-AFB1 with fixed concentration, and reacting with AFB1 monoclonal antibody on a functional sensing interface; 4) and detecting an electrochemiluminescence signal on a signal acquisition interface by using the working principle of the bipolar electrode. The invention utilizes BPE to convert the electrochemical signal of chemical reaction into electrogenerated chemiluminescence signal which can be sensitively detected, thus solving the problem that the electrochemistry can not distinguish Faraday current and charging current; the BPE is utilized to physically isolate the functional sensing interface from the signal acquisition interface, so that direct contact between an optically active molecule and a complex reaction system is avoided, the false positive phenomenon is effectively inhibited, the analysis and detection range is expanded, the detection method is simplified, and the method has the advantages of simplicity, convenience, sensitivity and good specificity.
Description
Technical Field
The invention belongs to the technical field of biosensing, and relates to a method for detecting aflatoxin B1 based on a visual BPE-ECL technology, in particular to a method for detecting aflatoxin B1 based on a visual BPE-ECL technology, which comprises the steps of functionally modifying a monoclonal antibody on the surface of gold nanoparticles at the cathode end of a screen-printed bipolar electrode, identifying and combining AFB1 or HRP-AFB1 by a competition method to cause the polymerization degree of aniline at the cathode end of the bipolar electrode to change, and observing the change of luminous intensity and luminous potential on an electrochemiluminescence analyzer so as to quantitatively detect AFB1 in agricultural products.
Background
Mycotoxins refer to toxic secondary metabolites produced during growth and reproduction of aspergillus, fusarium or penicillium, are various in variety, have been separated and identified at present, and comprise more than 400 species, such as aflatoxin, zearalenone/alcohol and the like, and can enter a food chain through contaminating grains and animal foods (such as milk, meat and eggs) fed by feeds contaminated by the mycotoxins. The food pollution caused by mycotoxin not only causes a great deal of food waste and serious food poisoning, but also poses a great threat to human survival and health due to the carcinogenic, teratogenic, mutagenic and other effects. Many mycotoxins are not easily removed during food processing due to their good physical, chemical and thermal stability. Meanwhile, the toxigenic fungi can pollute food in the whole food chain stage of growth, harvesting, storage, processing and even transportation of grains and fruits, and further generate mycotoxin. Contamination by mycotoxins is therefore considered an unavoidable and unpredictable problem and is of constant concern worldwide.
At present, the detection method of mycotoxin in food in China mainly comprises a biological identification method, a chemical analysis method, an immunoassay method and an instrumental analysis method. The biological identification method mainly comprises a seed germination test and a vomit test, but the method is not beneficial to rapid detection, only can verify a toxic part and a toxic mechanism, only can carry out qualitative analysis and is rarely adopted; the chemical analysis method mainly comprises thin layer chromatography, but the method has the disadvantages of complex operation process, large sample pretreatment workload, high danger and poor accuracy due to the fact that the standard substance needs to be directly contacted in the detection process; the immunoassay mainly comprises an ELISA method and a colloidal gold immunochromatography method, but the method has poor reproducibility, can not accurately quantify, is easy to generate false positive results, and has a plurality of experimental interference factors (temperature and reaction time); the instrumental analysis method mainly comprises high performance liquid chromatography, and the method has complex pretreatment and expensive experimental instruments, and cannot rapidly screen a large number of samples.
The bipolar electrode (BPE) and the external power supply are not connected by a lead, so that the construction of the detection device is simpler and more convenient. In addition, the BPE can physically isolate the reaction system from the signal measurement system, and an analyte does not need to participate in the Electrochemiluminescence (ECL) reaction of the anode, so that the direct contact of a photoactive molecule and a complex reaction system is avoided, and the application range of the ECL is greatly expanded. In addition, the BPE is easy to array, and ECL signals on each sensing interface are captured by a Charge Coupled Device (CCD) or a photomultiplier tube (PMT) to realize high-flux detection, so that the information quantity obtained in a single analysis can be improved. BPE signal amplification is better than that of a traditional three-electrode system, and high-sensitivity ECL detection can be realized by respectively modifying the anode and the cathode of the BPE signal. The perfect combination of BPE and ECL technology has many advantages, and is very suitable for analyzing and detecting mycotoxin in agricultural products with complex components.
Therefore, it is important to develop an advanced mycotoxin rapid and efficient detection method and to realize miniaturization, integration and multi-channel detection of a detection instrument.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a method for detecting aflatoxin B1 based on a visual BPE-ECL technology, wherein a functional sensing interface is constructed by using a competitive immunization method after the cathode end of a bipolar electrode is plated with gold, and the change of luminous intensity and luminous potential is observed on an electrochemiluminescence analyzer due to the presence or absence of AFB1 in a sample, so that the method for detecting aflatoxin B1 in agricultural products by using the visual BPE-ECL technology is established.
The technical scheme is as follows: in order to solve the technical problem, the invention provides a method for detecting aflatoxin B1 based on a visual BPE-ECL technology, which comprises the following steps:
1) preparing a screen printing bipolar electrode, wherein the bipolar electrode comprises a cathode end and an anode end;
2) constructing a functional sensing interface: gold plating the cathode end of the screen printing bipolar electrode prepared in the step 1), soaking in a sulfydryl-polyethylene glycol-carboxyl solution, and sealing at room temperature overnight; after cleaning, soaking a cathode in a mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide; after cleaning, adding AFB1 monoclonal antibody at the cathode end to construct a functional sensing interface;
3) mixing an AFB1 standard substance with a known concentration and an HRP-AFB1 with a fixed concentration, and reacting with an AFB1 monoclonal antibody on a functional sensing interface to construct a standard curve of the concentration and the luminous intensity of AFB 1;
4) mixing an AFB1 sample to be detected with unknown concentration with HRP-AFB1 with fixed concentration, reacting with an AFB1 monoclonal antibody on a functional sensing interface, and catalyzing aniline to polymerize in situ by the HRP assembled on the functional sensing interface to generate polyaniline;
5) detecting an electrochemiluminescence signal at the anode end of the screen printing bipolar electrode by using the working principle of the bipolar electrode to obtain luminous intensity, and obtaining the concentration of AFB1 in the sample to be detected by using the linear relation between the known concentration of AFB1 and the luminous intensity.
Wherein, the preparation steps of the screen printing bipolar electrode in the step 1) are as follows: firstly, selecting a polyethylene glycol terephthalate electric inert material as a substrate, and then printing two working electrode leads on two ends of the substrate by using printing ink to obtain a substrate; then drying the substrate, printing a carbon electrode between the two working electrode leads and drying to obtain a cathode end and an anode end of the bipolar electrode; then printing an electrode standard layer by adopting light-cured insulating paste and curing by using ultraviolet light; and finally, printing the electrode insulating layer by using the light-cured insulating paste and curing by using ultraviolet light. The whole screen-printed electrode is about 3cm long, about 1cm wide, and the bipolar electrode lead is about 12mm long.
Wherein, the construction steps of the functional sensing interface in the step 2) are as follows: adding a PBS buffer solution containing chloroauric acid at the cathode end of a bipolar electrode, adding the PBS buffer solution at the anode end, applying scanning voltage to the two ends of the screen printing bipolar electrode through an electrochemical workstation, gradually yellowing the cathode end along with the chloroauric acid obtaining electrons at the cathode end of the bipolar electrode, depositing gold nanoparticles at the cathode end, cleaning the prepared bipolar electrode with ultrapure water, and drying in the air; soaking the cathode end of the bipolar electrode in a sulfydryl-polyethylene glycol-carboxyl solution, and sealing at room temperature overnight; ③ after being cleaned by PBS, the cathode is soaked in the mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide for 2 hours; and fourthly, after PBS is cleaned, adding the AFB1 monoclonal antibody at the cathode end, and incubating for 2 hours at room temperature in a dark place.
Specifically, the step 3) of constructing the functional sensing interface includes the following steps: 20. mu.L of a 10 XPBS buffer solution containing 1% chloroauric acid was added to the cathode side of the bipolar electrode, and 20. mu.L of a 10 XPBS buffer solution was added to the anode side. And applying a certain scanning voltage (3.0V-6.0V) to the two ends of the screen printing bipolar electrode through the electrochemical workstation, wherein the cathode end of the bipolar electrode gradually turns yellow along with electrons obtained by chloroauric acid at the cathode end of the bipolar electrode, and the gold nanoparticles (AuNPs) are deposited at the cathode end. And cleaning the prepared bipolar electrode by using ultrapure water, and drying in the air. Soaking the cathode end of the bipolar electrode in 1mM sulfhydryl-polyethylene glycol-carboxyl (SH-PEG-COOH) solution, and sealing at room temperature overnight; ③ after being cleaned by PBS, the cathode end is soaked in a mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2h (room temperature and sealing); (iv) after PBS cleaning, 20. mu.L of 100ng mL was added to the cathode end-1The AFB1 monoclonal antibody was incubated at room temperature for 2 hours in the absence of light. At this point, the functional sensing interface (bipolar electrode cathode terminal) is constructed.
Wherein, the step 3) comprises the following steps: 10 uL of AFB1 with unknown concentration, 10 uL and 100ng mL-1The HRP-AFB1 mixed solution is dripped at the cathode end of the bipolar electrode, and incubated for 2 hours at room temperature in a dark place; ② after PBS cleaning, adding 20 μ L, 0.1M acetic acid/sodium acetate buffer solution (200mM aniline, 20mM hydrogen peroxide, 0.5 μ M DNA, pH 4.3) at the cathode end, and incubating for 2h at room temperature in the dark. The DNA is A59, particularly DNA with 59 bases a, and the function of the DNA is to provide a template for aniline polymerization.
Wherein, the step 4) comprises the following specific steps: adding a co-reactant at the anode end of the bipolar electrode, and detecting an electrochemiluminescence signal on a signal acquisition interface (the anode end of the bipolar electrode) by utilizing the working principle of the bipolar electrode: specifically, an electrochemiluminescence analyzer is used for applying a certain voltage to two ends of a screen printing bipolar electrode to detect an electrochemiluminescence signal of a signal sensing interface.
Wherein the co-reactant comprises 10mM Ru (bpy)3(Cl)2·6H2O and 50mM TPA.
The invention relates to a method for detecting AFB1 in agricultural products by using a visual BPE-ECL technology, which is characterized in that after the cathode end of a bipolar electrode is plated with gold, a functional sensing interface is constructed by using a competitive immunization method, and the change of luminous intensity and luminous potential observed on an electrochemiluminescence analyzer is caused by the presence or absence of AFB1 in a sample, so that the method for detecting the AFB1 in the agricultural products by using the visual BPE-ECL technology is established. The invention firstly fixes the antibody of aflatoxin B1(100ng/mL) to the cathode end of a bipolar electrode, then, the known concentration of HRP-labeled aflatoxin B1 is used for binding the antibody, and then the concentration gradient of aflatoxin B1 is added to compete with HRP-AFB1, as HRP can catalyze aniline polymerization to cause the change of luminous voltage and luminous signals, then the relation between AFB1 with different concentrations and luminous intensity is obtained to obtain a standard curve, and then AFB1 with unknown concentration and HRP-AFB1 with known fixed concentration in a sample to be detected are mixed, competes with the monoclonal antibody on the functional sensing interface for combination, and the HRP assembled on the interface catalyzes aniline to polymerize in situ to generate polyaniline which is an electrochemical active substance, thereby causing the change of oxidation-reduction potential, peak current and ECL intensity in an electrochemical system, and obtaining the concentration of AFB1 in the sample to be detected by using a standard curve.
Has the advantages that: compared with the prior art, the invention has the following characteristics and advantages:
(1) the bipolar electrode (BPE) and the external power supply are not connected by a lead, so that the construction of the detection device is simpler and more convenient.
(2) The BPE can physically isolate the reaction system from the signal measurement system, and an analyte does not need to participate in the Electrochemiluminescence (ECL) reaction of the anode, so that the direct contact of an optically active molecule and a complex reaction system is avoided, and the application range of the ECL is greatly expanded.
(3) BPEs are also easy to array, and use Charge Coupled Devices (CCDs) or photomultiplier tubes (PMTs) to capture ECL signals at each sensing interface for high-throughput detection can increase the amount of information obtained in a single assay.
(4) BPE signal amplification is better than that of a traditional three-electrode system, and high-sensitivity ECL detection can be realized by respectively modifying the anode and the cathode of the BPE signal.
Drawings
FIG. 1, an experimental flow chart showing a method of visualizing BPE-ECL technology for detecting AFB1 in agricultural products;
FIG. 2, shows a layout (A) of a screen printed bipolar electrode preparation, front side of the finished product (B left), back side of the finished product (B right), CV signal (C) for bare electrode, ECL signal (D) for bare electrode;
FIG. 3 shows SEM images (B) of cathode gold plating (A) of the bipolar electrode in the functional sensing interface construction, 0 (a), 2(B), 4 (C) and 6 (d) gold plating, and ECL (C) and C (C) insets of the bipolar electrode gold plating effect images of 0, 2, 4 and 6 gold plating;
fig. 4, experimental principle verification: cleaning without a template (A), cleaning with a template (B), not cleaning with a template (C), not cleaning with a template (D);
FIG. 5 shows the optimization of DNA concentration (A), aniline concentration (B), hydrogen peroxide concentration (C), and polymerization time (D);
FIG. 6, establishment of a standard curve; a graph (A) of voltage and electrochemiluminescence intensity, a graph (B) of concentration and electrochemiluminescence intensity, and a graph (C) of logarithmic concentration and electrochemiluminescence intensity corresponding to different concentrations of AFB 1;
figure 7, selectivity and specificity of the immunosensor to AFB 1. Detailed Description
The present invention is further illustrated by the following specific examples and the accompanying drawings, and it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.
Reagents and instruments used in this experiment:
aflatoxin B1 murine monoclonal antibody (mAb), aflatoxin B1-HRP (AFB1-HRP), aflatoxin B1-BSA (AFB1-BSA), chlorineGold acid (HAuCl)4·3H2O), ruthenium (II) trisbipyridyl chloride hexahydrate (Ru (bpy)3(Cl)2·6H2O), tri-n-propylamine (TPA), DNA (a59, specifically 59 base a DNA, which serves to provide a template for aniline polymerization.
Aniline, hydrogen peroxide (30%) hydrogen peroxide), mercapto-polyethylene glycol-carboxyl (SH-PEG-COOH), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), N-hydroxysuccinimide (NHS), electrochemical workstation (CHI750E), transmission electron microscope (JEM-2010, Hitachi, Japan), mixed vortexer (IKAGerman), centrifuge (eppendorf german), digital camera (Canon IXUS 115, Japan), MPI-E electrochemiluminescence analyzer (MPI-easalysis Client System)
Example 1
The method for detecting the AFB1 in the agricultural products based on the visual BPE-ECL technology comprises the following steps:
1. preparing a screen printing bipolar electrode: firstly, selecting a polyethylene terephthalate (PET) cheap electric inert material as a substrate, and then printing two working electrode leads at two ends of the substrate by using silver ink; then drying the substrate, printing carbon electrodes between the two working electrodes by using carbon paste slurry, and drying to obtain an anode and a cathode of the bipolar electrode; then, printing an electrode standard layer by using light-cured insulating paste (ultraviolet light-cured insulating ink and the like) and curing by using ultraviolet light; and finally, printing the electrode insulating layer by using the light-cured insulating paste and curing by using ultraviolet light. The whole screen-printed electrode is about 3cm long, about 1cm wide, and the bipolar electrode lead is about 12mm long. As shown in fig. 2, the bipolar electrode is shown on the front side in fig. 2(B) and on the back side in fig. 2 (B).
Principle verification step: adding 20 μ L of 1 × PBS into the anode and cathode of the bipolar electrode, connecting the bipolar electrode to an electrochemical workstation by a connector, and scanning the electrode by cyclic voltammetry, as shown in FIG. 2(C), which shows that under the action of a certain voltage, current is generated on the surface of the bipolar electrode, and shows that the prepared bipolar electrode has an oxidation-reduction loop; mu.L of 1 XPBS was added to the cathode side of the bipolar electrode and 20. mu.L of the co-reactant (10mM Ru (bpy)) was added to the anode side of the bipolar electrode3(Cl)2·6H 20, 50mM TPA), the bipolar electrode was connected to an electrochemiluminescence analyzer (ECL) by a connecting wire, as shown in fig. 2(D), indicating that a chemiluminescent signal was generated at the anode of the bipolar electrode under a certain voltage, which also indicates that the prepared bipolar electrode had a redox loop, which could be utilized in subsequent experiments.
2. The construction steps of the functional sensing interface are as follows: 20. mu.L of a 10 XPBS buffer solution containing 1% chloroauric acid was added to the cathode side of the bipolar electrode, and 20uL of 10 XPBS buffer solution was added to the anode side. And applying a certain scanning voltage (3.0V-6.0V) to the two ends of the screen printing bipolar electrode through the electrochemical workstation, wherein the cathode end of the bipolar electrode gradually turns yellow along with electrons obtained by chloroauric acid at the cathode end of the bipolar electrode, and the gold nanoparticles (AuNPs) are deposited at the cathode end. And cleaning the prepared bipolar electrode by using ultrapure water, and drying in the air. Soaking the cathode end of the bipolar electrode in 1mM sulfhydryl-polyethylene glycol-carboxyl (SH-PEG-COOH) solution, and sealing at room temperature overnight; ③ after being cleaned by PBS, the cathode end is soaked in a mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2h (room temperature and sealing); (iv) after PBS cleaning, 20. mu.L of 100ng mL of the solution was added to the cathode-1The AFB1 monoclonal antibody-aflatoxin B1 murine monoclonal antibody (mAb) was incubated at room temperature for 2h in the absence of light. At this point, the functional sensing interface (bipolar electrode cathode terminal) is constructed.
Principle verification step: adding 20 mu L of 10 multiplied by PBS buffer solution at the cathode end, adding 20 mu L of 10 multiplied by PBS buffer solution at the anode end, applying a certain scanning voltage (0V-6.0V) to the screen printing electrode by adopting a cyclic voltammetry method through an electrochemical workstation, wherein the scanning speed is 0.2V s-1The number of scanning turns is 20 turns, the sampling interval is 0.001V, and the electrode is cleaned; gold nanoparticles were then deposited on the cathode side of the closed BPE, 20. mu.L of PBS buffer containing 1% chloroauric acid was added on the cathode side, and 20. mu.L of 10 XPBS buffer was added on the anode side. A certain constant voltage (3.0V-6.0V) is applied to the screen printing electrode through the electrochemical workstation, and the cathode end of the bipolar electrode gradually turns yellow along with the electrons obtained by the chloroauric acid at the cathode end, which shows thatGold nanoparticles (AuNPs) were modified onto the cathode. The experimental procedure is shown in FIG. 3 (A). And cleaning the prepared bipolar electrode by using ultrapure water, and drying in the air. And the bare electrode and the gold-plated electrode are characterized by a scanning electron microscope, the gold-plating times are optimized, the density of gold nanoparticles is increased along with the increase of the gold-plating times, but the gold-plating times larger than 4 times can cause the nonuniformity of an AU film, which is not beneficial to the stable existence of the gold film, can influence the repeatability and is also not beneficial to the subsequent experiment (figure 3B). ECL signal measurements were then performed on the gold-plated electrode and the bare bipolar electrode anode, respectively (fig. 3C). Through the characterization of the surface of the gold-plated electrode and the measurement of an ECL signal, the fact that 4 times of gold plating can form a uniform gold film and has strong ECL strength is found, and therefore 4 times of gold plating are selected for carrying out subsequent experiments. The gold plated bipolar electrode is shown in the inset of fig. 3.
3. Verification of experimental principle and optimization of experimental conditions
After a standard substance (100ng/ml AFB1) of AFB1 and HRP-AFB1 with a fixed concentration are mixed, the mixture reacts with a monoclonal antibody on a functional sensing interface, and the HRP assembled on the interface catalyzes aniline to polymerize in situ to generate polyaniline, wherein the steps are as follows: 20 μ L (10 μ L of 100ng/mL AFB1 and 10 μ L, 100ng mL)-1HRP-AFB1) was added dropwise to the cathode end of the bipolar electrode and incubated at room temperature for 2h in the dark. ② after PBS cleaning, adding 20 μ L, 0.1M acetic acid/sodium acetate buffer solution (200mM aniline, 20mM hydrogen peroxide, 0.5 μ M DNA, pH 4.3) at the cathode end, and incubating for 2h at room temperature in the dark. ③ No clean, and 20. mu.L of a coreactant (10mM Ru (bpy)) was added to the anode3(Cl)2·6H2O, 50mM TPA), measured by connecting the bipolar electrode to an electrochemiluminescence analyzer (ECL) using a connector.
Principle verification step: after the 100ng/ml AFB1 and the HRP-AFB1 with a fixed concentration are mixed, the mixture reacts with a monoclonal antibody on a functional sensing interface, the HRP assembled on the interface catalyzes aniline to polymerize in situ to generate polyaniline, and whether the solution after aniline polymerization is cleaned and whether a DNA template has an influence on an experimental result (figure 4) is detected. As shown in fig. 4A, if no DNA was added during aniline polymerization and the solution was washed with PBS after the polymerization reaction, the experimental group and the blank group had a potential difference of 0.03V and only had an ECL intensity difference of about 100, which did not allow the experimental group and the blank group to be distinguished from each other; as shown in FIG. 4B, if DNA is added during aniline polymerization, but PBS is used for washing after the polymerization reaction, the experimental group and the blank group have a potential difference of 0.18V and only have an ECL intensity difference of about 1000, so that the experimental group and the blank group cannot be distinguished; as shown in FIG. 4C, if no DNA is added during the polymerization of aniline, but no washing is performed after the polymerization reaction, the experimental group and the blank group have a potential difference of 0.23V and an ECL strength difference of about 3000, and the experimental group and the blank group have a certain difference; as shown in FIG. 4D, if DNA is added during aniline polymerization but not washed after the polymerization reaction, the experimental group and blank group have a potential difference of 0.5V and an ECL intensity difference of about 10000, and the experimental group and blank group can be distinguished from each other; in conclusion, DNA with a certain concentration is added in the aniline polymerization process, and the mixture is not cleaned after the polymerization reaction, so that the experimental group and the blank group have larger potential and ECL strength difference, and the experimental group and the blank group can be effectively distinguished; then, the concentration of DNA, the concentration of aniline, the concentration of hydrogen peroxide and the time of polymerization reaction are respectively optimized; as shown in FIG. 5A, by adding DNA (0, 0.1, 0.5, 1.0, 5.0, 10. mu.M) of different concentrations to a sodium acetate buffer solution and measuring the potentials of the experimental group and blank group, respectively, the degree of polymerization of aniline was expressed by Δ E (potential difference between blank group and experimental group), it was found that the potential difference was the largest at 0.5. mu.M, so the optimum DNA concentration was 0.5. mu.M; as shown in FIG. 5B, the degree of aniline polymerization is represented by the intensity of the blank ECL by adding aniline (50, 100, 150, 200, 250mM) in different concentrations to the sodium acetate buffer solution, the ECL intensity gradually decreases with the increase of aniline concentration, and when the aniline concentration reaches 200mM, the ECL intensity does not change, indicating that the optimal aniline concentration is 200 mM; as shown in fig. 5C, hydrogen peroxide (0, 10, 20, 35, 50mM) with different concentrations was added into the sodium acetate buffer solution, the intensity of the blank ECL indicated the degree of aniline polymerization, and as the hydrogen peroxide concentration increased, the ECL intensity gradually decreased, and when the aniline concentration exceeded 20mM, the ECL intensity increased to some extent, indicating that the optimal hydrogen peroxide concentration was 20 mM; as shown in fig. 5D, the polymerization degree of aniline is represented by the intensity of the blank ECL through polymerization reactions (0, 15, 30, 45, 60, 90, 120, 240, 300min) at different times, the ECL intensity gradually decreases with the increase of the polymerization reaction time, and the ECL intensity does not change any more after the polymerization reaction time reaches 120min, which indicates that the optimal polymerization reaction time is 120 min; in conclusion, the optimal DNA concentration is 0.5. mu.M, the optimal aniline concentration is 200mM, the optimal hydrogen peroxide concentration is 20mM, and the optimal polymerization reaction time is 120 min.
EXAMPLE 2 establishment of Standard Curve- -determination of detection Limit and detection Range
Respectively taking 10 μ L of different concentrations (0, 0.1, 0.5, 1, 5, 10, 20, 50, 100ng mL)-1) The AFB1 standard substance is mixed with 10 mu L of 100ng mL-1HRP-AFB1 was mixed and allowed to react with the monoclonal antibody on the functional sensing interface constructed in example 1, and after incubation for 2 hours in a closed environment at room temperature, 20. mu.L of a 0.1M acetic acid/sodium acetate buffer (200mM aniline, 20mM hydrogen peroxide, 0.5. mu.M DNA, pH 4.3) was added to the cathode side after washing with PBS, and incubation for 2 hours at room temperature in the absence of light was carried out. No clean, 20. mu.L of a coreactant (10mM Ru (bpy)) was added to the anode side3(Cl)2·6H2O, 50mM TPA), measured by connecting the bipolar electrode to an electrochemiluminescence analyzer (ECL) using a connector. The results are shown in FIG. 6, when the concentration of AFB1 is 0.1-100ng mL-1The electrochemiluminescence intensity and the concentration of AFB1 show good linear relation, and the detection limit is 0.033ng mL-1The detection range is 0.1-100ng mL-1。
Example 3 specific detection
Adding HRP-AFB1 and different mycotoxin solutions into 7 EP tubes to obtain final concentration of 100ng mL-1HRP-AFB1 antigen and 100ng mL-1Mycotoxins, 7 tubes 100ng mL each-1HRP-AFB1 antigen and 10ng mL-1BSA-AFB1、100ng mL-1HRP-AFB1 antigen and 10ng mL-1Aflatoxin M1 (AFM1), 100ng mL-1HRP-AFB1 antigen and 100ng mL-1Zearalenone (ZEN), 100ng mL-1HRP-AFB1 antigen and 100ng mL-1Ochratoxins A (OTA), 100ng mL-1HRP-AFB1 antigen and 100ng mL-1Deoxynivalenol (DON) 100ng mL-1HRP-AFB1 antigen and 100ng mL-1mu.L of each of the Patulin (Patulin) was added to the functional sensor interface (cathode end of bipolar electrode) constructed in example 1, and after a competitive reaction for 2 hours, 20. mu.L of a 0.1M acetic acid/sodium acetate buffer solution (200mM aniline, 20mM hydrogen peroxide, 0.5. mu.M DNA, pH 4.3) was added to the cathode end after washing with PBS, and the mixture was incubated at room temperature in the dark for 2 hours. No clean, and 20. mu.L of a co-reactant (10mM Ru (bpy)) was added to the anode3(Cl)2·6H2O, 50mM TPA), measured by connecting the bipolar electrode to an electrochemiluminescence analyzer (ECL) using a connector. The experimental results are shown in fig. 7, and the immunosensor has good specificity and selectivity on AFB 1.
EXAMPLE 4 actual sample testing
According to the actual samples (rice, wheat, corn, sorghum, barley and buckwheat) processed by the enzyme-linked immunosorbent assay screening method of the Chinese national food safety standard (GB 2761-2017), firstly, at least 100g of samples are respectively weighed, a grinder is used for grinding the samples, and the ground samples pass through a test sieve with the aperture of 1 mm-2 mm. Respectively taking 5.0g of sample in a 50mL centrifuge tube, accurately adding 25.0mL of methanol-water (1: 1), shaking for 15min, filtering, discarding 1/4 primary filtrate, collecting sample filtrate, taking the sample filtrate as sample extract, and diluting with PBS (1: 1) in the sample extract to obtain sample liquid to be detected.
On the functional sensing interface constructed in example 1, 20. mu.L of the above-treated real sample (rice, wheat, corn, sorghum, barley, buckwheat) and 100ng mL of the solution were dropped-1After a competitive reaction was performed for 2 hours with a HRP-AFB1 mixed solution, after washing with PBS, 20. mu.L of a 0.1M acetic acid/sodium acetate buffer solution (200mM aniline, 20mM hydrogen peroxide, 0.5. mu.M DNA, pH 4.3) was added to the cathode, and the mixture was incubated at room temperature for 2 hours with exclusion of light. No clean, 20. mu.L of a coreactant (10mM Ru (bpy)) was added to the anode side3(Cl)2·6H2O, 50mM TPA), measured by connecting the bipolar electrode to an electrochemiluminescence analyzer (ECL) using a connector. According to the same pretreatment method, using ELISA and the detection method of the biosensor prepared by us, we are respectively right toThe six grains are subjected to an addition recovery test, and the designed biosensor is found to have higher recovery rate through comparison of the two methods, which indicates that the prepared sensor is more accurate and more reliable.
The results of the experiment are shown in table 1.
Table 1 comparison of recovery rates of biosensors and ELISA kits in grain samples.
aThe data in bold italics is the limit value of each grain sample in the mycotoxins in the food, the Chinese national food safety standard (GB 2761-.bFive independent measurements per data point.
Claims (7)
1. The method for detecting aflatoxin B1 based on the visual BPE-ECL technology is characterized by comprising the following steps: 1) preparing a screen printing bipolar electrode, wherein the screen printing bipolar electrode comprises a cathode end and an anode end; 2) constructing a functional sensing interface: gold plating the cathode end of the screen printing bipolar electrode prepared in the step 1), soaking in a sulfydryl-polyethylene glycol-carboxyl solution, and sealing at room temperature overnight; after cleaning, soaking a cathode in a mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide; after cleaning, adding AFB1 monoclonal antibody at the cathode end to construct a functional sensing interface; 3) mixing an AFB1 standard substance with a known concentration and an HRP-AFB1 with a fixed concentration, and reacting with an AFB1 monoclonal antibody on a functional sensing interface to construct a standard curve of the concentration and the luminous intensity of AFB 1; 4) mixing an AFB1 sample to be detected with unknown concentration with HRP-AFB1 with fixed concentration, reacting with an AFB1 monoclonal antibody on a functional sensing interface, and catalyzing aniline to polymerize in situ by the HRP assembled on the functional sensing interface to generate polyaniline; 5) detecting an electrochemiluminescence signal at the anode end of the screen printing bipolar electrode by using the working principle of the bipolar electrode to obtain luminous intensity, and obtaining the concentration of AFB1 in the sample to be detected by using the linear relation between the known concentration of AFB1 and the luminous intensity.
2. The method for detecting aflatoxin B1 based on the visual BPE-ECL technology as claimed in claim 1, wherein the step 1) of preparing the screen-printed bipolar electrode comprises the following steps: firstly, selecting a polyethylene glycol terephthalate electric inert material as a substrate, and then printing two working electrode leads at two ends of the substrate to obtain a substrate; then drying the substrate, printing a carbon electrode between the two working electrode leads and drying to obtain a cathode end and an anode end of the bipolar electrode; then printing an electrode standard layer and curing; and finally, printing an electrode insulating layer and curing to obtain the conductive film.
3. The method for detecting aflatoxin B1 based on the visualized BPE-ECL technology as claimed in claim 2, wherein the construction steps of the functional sensing interface in the step 2) are as follows: adding a PBS buffer solution containing chloroauric acid at the cathode end of a bipolar electrode, adding the PBS buffer solution at the anode end, applying scanning voltage to the two ends of the screen printing bipolar electrode through an electrochemical workstation, gradually yellowing the cathode end along with the chloroauric acid obtaining electrons at the cathode end of the bipolar electrode, depositing gold nanoparticles at the cathode end, cleaning the prepared bipolar electrode with ultrapure water, and drying in the air; soaking the cathode end of the bipolar electrode in a sulfydryl-polyethylene glycol-carboxyl solution, and sealing at room temperature overnight; ③ after being cleaned by PBS, the cathode is soaked in the mixed solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and N-hydroxysuccinimide; and fourthly, after PBS is cleaned, adding the AFB1 monoclonal antibody at the cathode end, and incubating at room temperature in a dark place to obtain the antibody.
4. The method for detecting aflatoxin B1 based on the visualized BPE-ECL technology as claimed in claim 1, wherein the specific steps in the step 4) are as follows: dripping mixed solution of AFB1 with unknown concentration and HRP-AFB1 with 100ngmL-1 at the cathode end of a bipolar electrode, and incubating for 2 hours at room temperature in a dark place; ② after PBS cleaning, adding acetic acid/sodium acetate buffer solution at the cathode end and incubating for 2h in the dark at room temperature.
5. The method for detecting aflatoxin B1 based on the visual BPE-ECL technique of claim 4, wherein the acetic acid/sodium acetate buffer solution comprises 200mM aniline, 20mM hydrogen peroxide, 0.5 μ MDNA, pH4.3, the DNA is A59, and the A59 is DNA of 59 bases a.
6. The method for detecting aflatoxin B1 based on the visualized BPE-ECL technology as claimed in claim 1, wherein the specific steps in the step 5) are as follows: adding a co-reactant at the anode end of the bipolar electrode, and detecting an electrochemiluminescence signal by utilizing the working principle of the bipolar electrode.
7. The method for detecting aflatoxin B1 based on the visual BPE-ECL technique of claim 6, wherein the co-reactants comprise 10mMRu (bpy)3(Cl) 2.6H 2O and 50 mMTPA.
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